Over the past 3-year grant period, the Program-Project on Cancer Gene Therapy on MSKCC has been entirely focused on the use of retroviral mediated gene transfer into hematopoietic cells for the purpose of (a) improving existing therapeutic protocols and (b) introducing new approaches to the control of malignant diseases. The main advances in this past 3-year period have been: In Project introducing new approaches to the control of malignant diseases. The main advances in this past 3- year period have been. In Project 1, achieving high efficiency transduction of murine and human T lymphocytes; and demonstrating in vitro and in vivo the sensitivity to gancyclovir of the transduced T lymphocytes. In Project 2, achieving high efficiency transduction of dendritic cells; and using them to generate cytotoxic T cells directed against ovalbumin peptides. In Project 3, producing new drug-resistant enzyme variants and demonstrating in a pre-clinical model that the use of genetically modified hematopoietic stem cells involves the survival of tumor-bearing mice treated with methotrexate. Over the next 5 years we plan to do the following. Project 1 will perfect methods for producing rapidly suicide-susceptible T cells targeted to EBV lymphoma and to ALL cells; clinical trials against these conditions will be undertaken. Project 2 will test the anti-tumor efficacy of dendritic cells expressing melanoma molecules first in a mouse model, and then in a clinical trial in patients with stage III/IV melanoma. In Project 3, a new major aim will be a clinical trial (currently at the stage of IRB approval) in patients with non-Hodgkin lymphoma, aiming to establish for how long hematopoietic cells expressing genes conferring resistance to methotrexate will persist in vivo, and to what extent drug administration will select immunoglobulin molecule and the surface receptor CD28 receive a co-stimulator signal from the tumor antigen. The PSMA molecule will be targeted in a pre- clinical model of human prostate carcinoma. The retroviral core will continue to support the development of new vectors for all 4 projects, and it will have a much expanded role in producing viral stocks for use in clinical trials; as well as taking responsibility for continuous monitoring of efficacy and safety. The administrative ore will continue to coordinate all 4 projects, and it will have an expanded role in providing data management for clinical trials in 3 projects and support in biostatistics for all. A third new core is being introduced (Imaging core),a imaging to provide state-of-the-art technology for in vivo imaging of the targeting to tumor sites of genetically engineered cells in mice and eventually in humans: preliminary data have been already obtained in work carried out with Project 1 and Project 4. Interactions among projects have been extensive and increasing particularly because optimization of vector design and transduction protocols accruing form one project have immediately benefitted others. In addition, the atmosphere of collaboration arising from regular joint meetings among investigators and fellows having similar aims has naturally produced valuable feedback on all projects. This in turn has helped in the formation of the present renewal proposal.